A novel, Golgi-Cox-based fluorescent staining method for visualizing full-length processes in primary rat neurons

• We established the Fluorescent Golgi method for cultured neurons.
• The method allows visualization of full-length processes of cultured neurons.
• Both the growth corns and spines of neurons were stained with high sensitivity.
• The key to visualization of this method is Fluorescent antibody is used.
• The advantages of our technique are simple procedure and stability of staining.

The Golgi method, a well-known method used for staining whole dendrites and axonal trees of neurons, has been used widely for studying dendritic growth in vivo. Although detailed structural examination of neurons and their processes stained by the Golgi method has elucidated the complicated neuronal circuit, application of the method in cultured neurons has been unsuccessful to date.Here, we report the development of a stable, highly sensitive Golgi-Cox method that allows visualization of full-length processes, including the dendritic spines and the growth cones, of cultured rat neurons. This modified staining method requires: (1) rat cultured neurons fixed with a mixture containing 4% paraformaldehyde and 12.5% glutaraldehyde before impregnation with mercury; (2) rapid freezing of the fixed neurons using dry ice; and (3) immersion of the fixed neurons in Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin-G antibody (Molecular Probes, Eugene, OR) for visualization after impregnation.